Mary L. Alpaugh

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cells sensitivity to Hsp90 inhibition.

Cooperative role of E-cadherin and sialyl-Lewis X/A-deficient MUC1 in the passive dissemination of tumor emboli in inflammatory breast carcinoma

Inflammatory breast carcinoma (IBC) is characterized by florid tumor emboli within lymphovascular spaces termed lymphovascular invasion (LVI). Using a human-scid model of IBC (MARY-X), we have demonstrated using retrovirally-mediated dominant-negative E-cadherin mutant approaches (H-2Kd-E-cad), that the tumor cell embolus (IBC spheroid) forms on the basis of an intact and overexpressed E-cadherin/α, β-catenin axis which mediates tumor cell–tumor cell adhesion analogous to the embryonic blastocyst and accounts for the compactness of the embolus. The tumor cell embolus (IBC spheroid), in contrast, fails to bind the surrounding vascular endothelial cells both in vitro and in vivo because of markedly decreased sialyl-Lewis X/A carbohydrate ligand-binding epitopes on its overexpressed MUC1 which are necessary for binding endothelial cell E-selectin. This tumor cell-endothelial cell aversion further contributes to the compactness of the IBC spheroid and its passivity in metastasis dissemination. This passivity is manifested by a dramatic increase in metastatic pulmonary emboli following palpation of the primary tumor. In assessing this passivity of metastatic dissemination, we compared the effects of palpation on MARY-X with the effects of palpation on a derived dominant-negative E-cadherin mutant (H-2Kd-E-cad), as well as other well known human tumoral xenografts exhibiting no (MCF-7, T47D), low (MDA-MB-231, MDA-MB-468) or high (C8161, M24met) levels of spontaneous metastasis but no LVI. Palpation of each xenograft similarly increased intratumoral pressure by 200% (10→30 mmHg) but dramatically increased the numbers and sizes of pulmonary metastases 10–100-fold (P<0.001) only in MARY-X. The mechanism of this effect was through an immediate post-palpation release of circulating tumor emboli detected 2–3 min after palpation (P<0.01) by human cytokeratin 19 RT–PCR of extracted RNA from 300 μl of murine blood. Although circulating human tumor cell–derived growth factors (IGF-I, IGF-II, TGF-α and TGF-β) and angiogenic factors (VEGF and bFGF) were detected by ELISA in murine serum of MARY-X, palpation did not further increase the circulating levels of these factors (P>0.1). Our findings support the cooperative role of E-cadherin and sialyl-Lewis X/A-deficient MUC1 in the passive dissemination of tumor emboli in IBC.

The epichaperome is an integrated chaperome network that facilitates tumour survival

Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes—dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically ‘rewired’ to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.

Synthesis of purine-scaffold fluorescent probes for heat shock protein 90 with use in flow cytometry and fluorescence microscopy

Fluorescent ligands for the heat shock protein 90 (Hsp90) were synthesized containing either fluorescein isothiocyanate (FITC), 4-nitrobenzo[1,2,5]oxadiazole (NBD) or the red shifted dye sulforhodamine 101 (Texas Red) conjugated to PU-H71. Two of the compounds, PU-H71-FITC2 (9) and PU-H71-NBD1 (8), were shown to be suitable for fluorescence-activated flow cytometry and fluorescence microscopy. Thus these molecules serve as useful probes for studying Hsp90 in heterogeneous live cell populations.

Breast Cancer-Derived Extracellular Vesicles: Characterization and Contribution to the Metastatic Phenotype

The study of extracellular vesicles (EVs) in cancer progression is a complex and rapidly evolving field. Whole categories of cellular interactions in cancer which were originally presumed to be due solely to soluble secreted molecules have now evolved to include membrane-enclosed extracellular vesicles (EVs), which include both exosomes and shed microvesicles (MVs), and can contain many of the same molecules as those secreted in soluble form but many different molecules as well. EVs released by cancer cells can transfer mRNA, miRNA, and proteins to different recipient cells within the tumor microenvironment, in both an autocrine and paracrine manner, causing a significant impact on signaling pathways, mRNA transcription, and protein expression. The transfer of EVs to target cells, in turn, supports cancer growth, immunosuppression, and metastasis formation. This review focuses exclusively on breast cancer EVs with an emphasis on breast cancer-derived exosomes, keeping in mind that breast cancer-derived EVs share some common physical properties with EVs of other cancers.

Abstract 3029: Biochemical evidence towards the existence of an oncogenic Hsp90 complex

To maintain homeostasis, cells employ intricate molecular machineries comprised of thousands of proteins programmed to execute well-defined functions. Dysregulation of these pathways, through protein mis-expression or mutation, provides biological advantages that confer the malignant phenotype. At the molecular level, this requires cells to invest energy in maintaining the stability and function of these proteins, and for this reason cancer cells co-opt molecular chaperones, including Hsp90. Hsp90 is recognized to play important roles in maintaining the transformed phenotype - the chaperone and its associated co-chaperones assist in the correct folding of cellular proteins, collectively referred to as “client proteins,” many of which are effectors of signal transduction pathways controlling cell growth, differentiation, the DNA damage response, and cell survival. Tumor cell addiction to these proteins (i.e. through mutations, aberrant expression, improper cellular translocation, etc) thus makes them critically reliant on Hsp90. While Hsp90 is expressed in all cells and tissues, it was shown that tumors preferentially contain Hsp90 that is in a higher order multi-chaperone complex with high affinity for certain Hsp90 inhibitors, while normal tissues harbor a latent, uncomplexed Hsp90 that has low affinity for these inhibitors. We here extend this model and propose that Hsp90 forms biochemically distinct complexes in cancer cells. In this view, a major fraction of cancer cell Hsp90 retains “house keeping” chaperone functions similar to normal cells, whereas a functionally distinct Hsp90 pool enriched or expanded in cancer cells specifically interacts with oncogenic proteins required to maintain tumor cell survival. Perhaps this Hsp90 fraction represents a cell stress specific form of chaperone complex that is expanded and constitutively maintained in the tumor cell context. Our data suggest that it may execute functions necessary to maintain the malignant phenotype. One such role is to regulate the folding of mutated (i.e. mB-Raf) or chimeric proteins (i.e. Bcr-Abl). We here also present experimental evidence for an additional role; that is, to facilitate scaffolding and complex formation of molecules involved in aberrantly activated signaling complexes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3029. doi:1538-7445.AM2012-3029

Ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics.

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Abstract A01: Pharmacological approaches to metastatic disease using an Inflammatory Breast Cancer model

Goal of Study: The lymphovascular embolus is a marker of breast cancer aggressiveness, recurrence, metastatic progression and therapeutic failure. This is especially reflective in inflammatory breast cancer (IBC) which presents as florid intravasation in situ of the lymphovasculature by tumor emboli. This study utilizes a novel 3-dimensional (3D) in vitro and in vivo model of IBC to systematically and strategically develop pharmacological approaches in the treatment of both IBC and metastatic disease. Background: Intravasation, the rate-limiting step of metastasis, is extremely important in tumor progression and expansion, yet the mechanism underlying intravasation is still poorly understood. The consequence of intravasation is the resultant lymphovascular embolus – the key defining feature of metastasis. This is most prevalent in IBC, the most lethal form of breast cancer. Clinical IBC features, erythema, peau d9orange (dimpling of thickened skin) and warmth are due to the IBC signature phenotype, namely extensive intravasation in situ of the lymphatic and blood vessels by tumor emboli. Nearly 100% of all women with IBC have lymph node involvement and approximately 25% of patients with IBC have distant metastases at diagnosis. The IBC xenograft model, MARY-X, has precisely captured the IBC phenotype, where the tumor emboli grow exclusively within the murine lymphatic and blood vessels and exhibit erythema of the overlying skin. MARY-X is an invaluable preclinical IBC model and an overall model of metastasis providing a means to validate and translate therapeutic strategies. MARY-X, in vitro, is a cellular derivative of primary tumor explants. These tumor cells form tight, compact aggregates of cells termed “MARY-X spheroids”. Comparable to human IBC emboli, a persistent, over-expression of an intact E-cadherin/α, β-catenin axis mediates the compaction of both in vitro and in vivo MARY-X spheroids and tumor emboli, respectively. These comparative architectural features (i.e. mimics the in vivo metastasis) of the spheroid of MARY-X provide an in vitro model with tractable in vivo applications. Research Strategy: By exploiting the MARY-X in vitro 3-D model, high throughput (HTP) screening is performed to systematically probe the factors involved in maintenance of tumor emboli architecture as it pertains to sensitivity/resistance to therapeutics in an overall effort to develop efficacious therapeutics for treatment of IBC and metastatic disease. Additionally, MARY-X spheroids co-cultured with bone marrow derived cells (BMDCs) in matrigel overlays are employed to assess targeting of mediators of crosstalk between tumor spheroid/embolus soluble factors (e.g. cytokines, exosomes) and the microenvironment. Soluble factors identified as mediators (i.e. targets) of metastatic progression, that showed the most promise in the HTP screen, are further validated in the in vivo preclinical model, MARY-X. A significant decrease in vessel density of the MARY-X tumor will be indicative of successful blocking of this ‘crosstalk’ i.e. lymphovasculogenesis or the formation of lymphovascular emboli (i.e. intravasation) – the defining feature of metastatic disease. Preliminary Data and Conclusions: Preliminary data show that dissolution of the spheroid/embolus augment apoptotic effects of chemo/irradiation therapy. Suggesting that apoptosis resistance of lymphovascular emboli may be independent of traditional apoptotic mechanisms. Therefore, therapy evasion, in part, is due to formation and maintenance of the architectural features of the lymphovascular embolus. In addition, abrogation of ‘crosstalk’ has proven to hinder the establishment of lymphovascular emboli. Citation Format: Mary L. Alpaugh, Emmanuel Theodorakis. Pharmacological approaches to metastatic disease using an Inflammatory Breast Cancer model. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A01.

Abstract 5598: A comprehensive study to analyze tumor sensitivity to HSP90 inhibition therapy.

There have been great advances in receptor-targeted breast cancer therapeutics. However, limited sensitivity and tumor resistance remain significant problems for many patients. To overcome these obstacles our laboratory has focused on targeting HSP90, a global mediator of oncogenic pathways involved in nearly all cancer types. Our laboratory has developed PU-H71 which selectively inhibits oncogenic HSP90 and is one of the first targeted therapies to show preclinical potent activity in the treatment of triple negative breast cancers. Here, we investigate how the complex biology of different types of breast tumors influences response to HSP90 inhibition. Our preliminary data indicate that sensitivity to PU-H71 goes beyond a tumor9s receptor status and is highly driven by its intracellular protein composition. Our objective is to identify specific pharmacodynamic changes in select HSP90 onco-client proteins and regulators of apoptosis associated with HSP90 inhibition as a tool to monitor therapeutic response. Using several different breast cancer cell lines we evaluate how alterations in specific onco-client phosphorylated species as well as the induction of pro-apoptotic protein cleavage correlates to a tumor9s sensitivity to HSP90 inhibition. In addition to testing cell lines, we have developed a system to investigate PU-H71 on primary tumors by treating surgery specimens ‘ex vivo’ (fresh tissue sectioning) to determine response and pharmacodynamic alterations in patient samples. Our long term goal is to establish specific pharmacodynamic changes (PD markers) that correlate to tumor sensitivity allowing us to better predict those patients more likely to benefit from HSP90 inhibition i.e. PU-H71 therapy. Citation Format: Matthew Riolo, Shanu Modi, Adriana Corben, Clifford Hudis, Mary Alpaugh, Gabriela Chiosis. A comprehensive study to analyze tumor sensitivity to HSP90 inhibition therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5598. doi:10.1158/1538-7445.AM2013-5598

Abstract 1263: Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1263. doi:1538-7445.AM2012-1263